In order to understand the molecular basis of diseases, particularly those involving single amino acid substitutions, we need to elucidate the relationship between protein structure and function at the molecular level. Therefore, the long-term goals of this project are to acquire a deeper understanding of the relationship between protein structure and function by using alkaline phosphatase as a model system. The function of this enzyme is to catalyze the nonspecific hydrolysis of phosphate esters, and the lack of the activity of this enzyme results in the fatal hereditary disease hypophosphatasia, which is due to insufficient phosphate for bone calcification. Alkaline phosphatase from mammals is closely related to the corresponding bacterial enzyme, and the enzyme from Escherichia coli has become the model for the study of all alkaline phosphatases. The specific aims of this proposal are to answer fundamental questions concerning the relationship between the structure and function of alkaline phosphatase. We will concentrate on the molecular details of the catalytic mechanism, the need to maintain a dimeric structure for the correct function of the enzyme, the mode by which information is passed between the subunits, a molecular explanation of intergenic complementation, the factors critical for correct secondary structure formation, the function of the metals in catalysis, and the contribution of the electrostatic field around the active site for catalysis. In order to accomplish this goal, we will take advantage of a set of thousands of point mutations created within the alkaline phosphatase gene more than 30 years ago in an early effort to prove that the gene and the protein produced from it were colinear. This set of mutants today provides a unique resource for the investigation of the interrelationship between the structure and function of alkaline phosphatase. Analysis of this set of mutants as well as mutants created during the last grant period will be accomplished by a variety of techniques including x-ray crystallography, 31P and 113Cd NMR, 18(O) isotope effects, and kinetic studies with phosphonates, phosphorothioates and the chiral (Rp) [O16,O17, O18] p-nitrophenyl phosphate. Correlations will be made between the functional changes induced by the amino acid substitution and the three-dimensional structure of the mutant enzymes. This work will not only be important for the understanding of this particular system, but more importantly for formulating general concepts about enzyme catalysis, and the function of metals in proteins, and providing a molecular explanation for intergenic complementation.